This invention relates to culture media, devices and methods for detecting and enumerating microorganisms in a sample, and particularly relates to media, devices and methods that employ ballasted pH indicators.
Methods of detecting microorganism growth using pH indicators have long been known. The presence of microorganisms is usually detected by a visual color or fluorescence change of the pH indicator in a zone surrounding the growing microbial colony that is caused by the reaction of the indicator with acid produced by the growing microorganisms.
A common problem when using pH indicators is that as the growing microorganisms continue to produce acid, the colored or fluorescent indicator zones increase in size and may begin to merge and overlap with the indicator zones of the surrounding nearby colonies. After a typical 18 to 24 hour incubation period, the adjacent indicator zones typically diffuse together, thereby making it difficult to differentiate between and enumerate the individual indicator zones which correspond to individual colonies.
The use of pH indicators at very high concentrations has recently been shown to decrease the detection time for coliform bacteria in a culture medium without deleteriously affecting bacterial growth. However, even with a decreased detection time, large numbers of colonies may prevent an observer from being able to distinguish between colonies that produce acid and those that do not produce acid, because the indicator zones may increase in size and diffuse together during incubation.
Another potential problem when selecting pH indicators is that some indicators that demonstrate good indicating and/or spectral properties are toxic to microbiological growth.
There is a need in the art for media that overcomes the deficiencies of the prior art that limit the spread of indicator zones when using pH indicators. In particular, there is a need for media, devices and methods of use thereof that control the spread of indicator zones when using pH indicators.
In one aspect of the present invention, a culture medium includes a medium for growing microorganisms and at least one ballasted pH indicator. The media has growth nutrients for the targeted microorganisms or growth nutrients may be added for use. Preferably, the medium is a cold water soluble powder. A variety of pH indicators may be used in the present invention, including potentially some indicators normally toxic to microbiological growth. The indicators include monoazo dyes; polyazo dyes; amino-, hydroxy-, and aminohydroxy- arylmethanes, including di- and tri-arylmethanes; modified phenolphthaleins; modified sulfonphthaleins; anthroquinones; xanthenes; polycylic aromatics; and naphthofluoresceins. Preferred ballasts include high molecular weight substances selected from the group consisting of cellulose; modified celluloses; polyvinyl alcohol (PVA); dextrans; amino-modified dextrans; guar gums; modified guar gums; xanthan gum; locust bean gum; and other polycarbohydrate gums.
In another aspect of the present invention, a device for detection and/or enumeration of microorganisms includes a culture medium as described above. A preferred device comprises a self-supporting waterproof substrate, an optional foam spacer, and a cover.
In still another aspect of the present invention, a method of detecting and enumerating microorganisms in a sample includes the steps of adding a sample suspected of including microorganisms to a culture medium containing a ballasted pH indicator, growing the microorganisms in the presence of the culture medium, and detecting a color or fluorescence change of the ballasted pH indicator in the medium.
The present invention overcomes deficiencies of the prior art by controlling the spreading of colored or fluorescent indicator zones throughout a culture medium. Control of the colored or fluorescent indicator zones is achieved by binding the pH indicator to a ballast. Surprisingly, these ballasted pH indicators control the spreading of the indicator zones in the culture medium without affecting the acid production capability of colonies in the medium. As a result, the merging of adjacent indicator zones is reduced or eliminated and differentiation between individual indicator zones may be enhanced even after an extended incubation time.
The present invention also provides media, devices and methods that may permit the use of pH indicators which normally would be considered toxic to microbiological growth. By ballasting the pH indicators, the indicators may not exhibit toxicity to the microorganisms.